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Saccharomyces boulardii (S. boulardii) is a fungal probiotic used to treat digestive disorders. However, the mechanism(s) by which S. boulardii affects the small intestine remains unclear. Here, we aimed to explore the effects of S. boulardii on the small intestine and the underlying mechanisms in mice with loperamide-induced constipation. While S. boulardii administration did not fully reverse the alterations in loperamide-induced defecation parameters, it altered the small intestinal floral composition toward a community conducive to alleviate constipation. Moreover, S. boulardii up-regulated the expression of tyrosine-protein kinase Kit (c-Kit), aquaporin 3 (AQP3), interleukin (IL)-10, myosin light chain kinase (MLCK), and phosphorylated myosin light chain 20 (P-MLC20), while concurrently down-regulating the expression levels of inducible nitric oxide synthase (iNOS), p65, and IL-17 A. These alterations indicate a discernible effect of small intestinal water reabsorption, inflammatory factor levels, and smooth muscle contraction. Saccharomyces boulardii also positively regulated small intestinal metabolite levels, such as fructose 6-phosphate, dihomo-alpha-linolenic acid, and 3-(4-hydroxyphenyl) lactate, and participated in metabolic pathways such as arginine biosynthesis, linoleic acid metabolism, and protein digestion and absorption. While not fully reversing defecation changes, Saccharomyces boulardii alters intestinal flora, up-regulates key proteins affecting water reabsorption and inflammation, and positively influences metabolic pathways. Our study provides serves as a basis for further studies on the application of S. boulardii in the treatment of intestinal disorders., Competing Interests: Declarations. Ethics approval and consent to participate: All experimental protocols were approved by the Animal Ethics Committee of People’s Liberation Army General Hospital, Beijing, China (2022-X18-71). Patient consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The effect of baicalin methyl ester (BME) on the regulation of mice intestinal barrier in the inflammatory response was studied in vivo and in vitro. Thirty six C57/BL mice were randomly divided into six groups (n = 6): control group; LPS group (LPS 3.5 mg/kg given intraperitoneal [ip] on day 7 of the study only), PBS group, and three BME groups (low: 50 mg/kg; medium: 100 mg/kg; high: 200 mg/kg) orally dosed with BME for 7d and LPS ip on day 7. All mice were sacrificed on day 8, and jejunum tissue collected for histopathology (H&E and PAS staining), protein expression of pro-inflammatory factors (TNF-α, IL-6, IL-8, IFN-γ) by ELISA, and intestinal tight junction proteins (ZO-1, occludin, claudin-1 and claudin-4) by Western Blot. Compared with the control group, LPS significantly increased the serum cytokines DAO (p < 0.01) and DLA (p < 0.01), upregulated the expression of pro-inflammatory factors, MLCK proteins (p <0.05) and increased the MLCK/ZO-1ratio (p <0.001). LPS also decreased the expression of claudin-4 (p < 0.01) in the jejunum and induced an inflammatory response damaging the jejunal mucosal barrier. Pretreatment with BME (100-200 mg/kg) significantly decreased the cytokines DAO (p < 0.05) and DLA (p < 0.01) in the serum, pro-inflammatory factors in the jejunum, significantly down-regulated the expression of MLCK (p <0.05) and the ratio of MLCK/ZO-1(p <0.001) but upregulated the expressions of ZO-1(p < 0.01), occludin (p < 0.05), claudin-1(p < 0.05) and claudin-4 (p < 0.05), and thereby restored the intestinal tissue structure, suggestive of alleviation of LPS-induced intestinal inflammation by BME. In vitro, MODE-K cells (derived from mice intestinal epithelium) were exposed to BME at 0 (control group-No LPS), 10, 20 and 40 μM BME for 24 h prior to LPS addition at 50 μg/mL for 2 h. LPS significantly increased the expression of pro-inflammatory factors, MLCK (p < 0.01) and the ratio of MLCK/ZO-1(p <0.001), decreased the expressions of ZO-1 (p < 0.05), occludin (p < 0.01), claudin-1 (p < 0.01) and claudin-4 (p < 0.01) in MODE-K cells compared with the control group. Compared with the LPS group, BME (10 - 40 μM) significantly decreased the expression of pro-inflammatory factors, MLCK (p < 0.05) and the ratio of MLCK/ZO-1(p <0.01) but increased the expressions of ZO-1(p < 0.01), occludin (p < 0.05) and claudin-4(p < 0.01) indicating an up-regulation of the expression of tight junction proteins by BME. On addition of extrinsic TNF-α plus LPS, the TNF- α level increased (p < 0.001) in MODE-K cells and the protein expression of MLCK (p < 0.01) was markedly up-regulated. Molecular docking predicted BME interacted with P65 by forming hydrogen bonds. IP-WB further confirmed that BME was directly bound to P65 protein in MODE-K cells. In conclusion, BME was able to restore the intestinal barrier through the P65 / TNF-α / MLCK / ZO-1 signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Overexpression or activation of oncogenes or loss of tumor-suppressor genes can induce cellular senescence as a defense mechanism against tumor development, thereby maintaining cellular homeostasis. However, cancer cells can circumvent this senescent state and continue to spread. Myosin light chain kinase (MLCK) is downregulated in many breast cancers. Here we report that downregulation of MLCK in normal breast epithelial cells induces a senescence-associated secretory phenotype and stimulates migration. The reduction of MLCK results in increased p21 Cip1 expression, dependent on p53 and the AKT-mammalian target of rapamycin pathway. Subsequently, p21 Cip1 promotes the secretion of soluble ICAM-1, IL-1α, IL-6 and IL-8, thereby enhancing collective cell migration in a non-cell-autonomous manner. These findings provide new mechanistic insights into the role of MLCK in cellular senescence and cancer progression., (© 2024. The Author(s).)

Deciphering the functional relevance of every protein is crucial to developing a better (patho)physiological understanding of human biology. The discovery and use of quality chemical probes propel exciting developments for developing drugs in therapeutic areas with unmet clinical needs. Myosin light-chain kinase (MLCK) serves as a possible therapeutic target in a plethora of diseases, including inflammatory diseases, cancer, etc. Recent years have seen a substantial increase in interest in exploring MLCK biology. However, there is only one widely used MLCK modulator, namely, ML-7, that too with a narrow working concentration window and high toxicity profile leading to limited insights. Herein, we report the identification of a potent and highly selective chemical probe, Myokinasib-II, from the synthesis and structure-activity relationship studies of a focused indotropane-based compound collection. Notably, it is structurally distinct from ML-7 and hence meets the need for an alternative inhibitor to study MLCK biology as per the recommended best practices. Moreover, our extensive benchmarking studies demonstrate that Myokinasib-II displays better potency, better selectivity profile, and no nonspecific interference in relevant assays as compared to other known MLCK inhibitors.

Objective: To investigate the mechanism of the protective effect of modified Pulsatilla decoction (, MPD) on the mechanical barrier of the ulcerative colitis (UC) intestinal epithelium in vitro and in vivo ., Methods: We established an intestinal epithelial crypt cell line-6 cell barrier injury model by using lipopolysaccharide (LPS). The model was then treated with p38 mitogen-activated protein kinase-myosin light chain kinase (p38MAPK-MLCK) pathway inhibitors, p38MAPK-MLCK pathway silencing genes (si-p38MAPK, si-NF-κB, and si-MLCK), and MPD respectively. Transepithelial electronic resistance (TEER) measurements and permeability assays were performed to assess barrier function. Immunofluorescence staining of tight junctions (TJ) was performed. In in vivo experiment, dextran sodium sulfate-induced colitis rat model was conducted to evaluate the effect of MPD and mesalazine on UC. The rats were scored using the disease activity index based on their clinical symptoms. Transmission electron microscopy and hematoxylin-eosin staining were used to examine morphological changes in UC rats. Western blotting and real-time quantitative polymerase chain reaction were performed to examine the gene and protein expression of significant differential molecules., Results: In in vitro study, LPS-induced intestinal barrier dysfunction was inhibited by p38MAPK-MLCK pathway inhibitors and p38MAPK-MLCK pathway gene silencing. Silencing of p38MAPK-MLCK pathway genes decreased TJ expression. MPD treatment partly restored the LPS-induced decreased in TEER and increase in permeability. MPD increased the gene and protein expression of TJ, while down-regulated the LPS-induced high expression of p-p38MAPK and p-MLC. In UC model rats, MPD could ameliorate body weight loss and disease activity index, relieve colonic pathology, up-regulate TJ expression as well as decrease the expression of p-p38MAPK and p-MLC in UC rat colonic mucosal tissue., Conclusions: The p38MAPK-MLCK signaling pathway can affect mechanical barrier function and TJ expression in the intestinal epithelium. MPD restores TJ expression and attenuates intestinal epithelial barrier damage by suppressing the p38MAPK-MLCK pathway.

Background: High doses of selenium are associated with heart disease prevalence in high-risk areas. Cardiac myosin light chain kinase (cMLCK) is an essential enzyme for normal function of heart tissue. Therefore, we studied the effect of high doses of selenium on the expression of cMLCK gene and its protein in normal heart tissue in rats., Materials and Methods: Twenty male rats were randomly divided into four groups: control, Se 0.3mg/kg, Se 1.5mg/kg, and Se 3mg/kg. Sodium-selenite was administered orally into drinking water for 20 weeks. Se levels of heart tissue were measured by atomic absorption. Serum creatine phosphokinase (CPK) and total serum antioxidant capacity were measured. Moreover, the concentration of MLCK protein and the gene expression level of cMLCK in normal heart tissue were analyzed., Results: Excess Se in dietary can significantly increase CPK. Se concentration of heart tissue in the Se 3mg/kg group was significantly higher than the control. cMLCK mRNA levels were decreased by 0.3mg/kg and 3mg/kg sodium selenite intake. There was no significant difference between the three groups for total antioxidant capacity and MLCK protein., Conclusion: High concentrations of selenium can probably effect on normal function of the heart tissue by changing the expression levels of cMLCK., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bifidobacterium bifidum strain BB1 causes a strain-specific enhancement in intestinal epithelial tight junction (TJ) barrier. Tumor necrosis factor (TNF)-α induces an increase in intestinal epithelial TJ permeability and promotes intestinal inflammation. The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α-induced increase in intestinal TJ permeability and to unravel the intracellular mechanisms involved. TNF-α produces an increase in intestinal epithelial TJ permeability in Caco-2 monolayers and in mice. Herein, the addition of BB1 inhibited the TNF-α increase in Caco-2 intestinal TJ permeability and mouse intestinal permeability in a strain-specific manner. BB1 inhibited the TNF-α-induced increase in intestinal TJ permeability by interfering with TNF-α-induced enterocyte NF-κB p50/p65 and myosin light chain kinase (MLCK) gene activation. The BB1 protective effect against the TNF-α-induced increase in intestinal permeability was mediated by toll-like receptor-2/toll-like receptor-6 heterodimer complex activation of peroxisome proliferator-activated receptor γ (PPAR-γ) and PPAR-γ pathway inhibition of TNF-α-induced inhibitory kappa B kinase α (IKK-α) activation, which, in turn, resulted in a step-wise inhibition of NF-κB p50/p65, MLCK gene, MLCK kinase activity, and MLCK-induced opening of the TJ barrier. In conclusion, these studies unraveled novel intracellular mechanisms of BB1 protection against the TNF-α-induced increase in intestinal TJ permeability. The current data show that BB1 protects against the TNF-α-induced increase in intestinal epithelial TJ permeability via a PPAR-γ-dependent inhibition of NF-κB p50/p65 and MLCK gene activation., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The global incidence of cardiac diseases is increasing, imposing a substantial socioeconomic burden on healthcare systems. The pathogenesis of cardiovascular disease is complex and not fully understood, and the physiological function of the heart is inextricably linked to well-regulated cardiac muscle movement. Myosin light chain kinase (MLCK) is essential for myocardial contraction and diastole, cardiac electrophysiological homeostasis, vasoconstriction of vascular nerves and blood pressure regulation. In this sense, MLCK appears to be an attractive therapeutic target for cardiac diseases. MLCK participates in myocardial cell movement and migration through diverse pathways, including regulation of calcium homeostasis, activation of myosin light chain phosphorylation, and stimulation of vascular smooth muscle cell contraction or relaxation. Recently, phosphorylation of myosin light chains has been shown to be closely associated with the activation of myocardial exercise signaling, and MLCK mediates systolic and diastolic functions of the heart through the interaction of myosin thick filaments and actin thin filaments. It works by upholding the integrity of the cytoskeleton, modifying the conformation of the myosin head, and modulating innervation. MLCK governs vasoconstriction and diastolic function and is associated with the activation of adrenergic and sympathetic nervous systems, extracellular transport, endothelial permeability, and the regulation of nitric oxide and angiotensin II. Additionally, MLCK plays a crucial role in the process of cardiac aging. Multiple natural products/phytochemicals and chemical compounds, such as quercetin, cyclosporin, and ML-7 hydrochloride, have been shown to regulate cardiomyocyte MLCK. The MLCK-modifying capacity of these compounds should be considered in designing novel therapeutic agents. This review summarizes the mechanism of action of MLCK in the cardiovascular system and the therapeutic potential of reported chemical compounds in cardiac diseases by modifying MLCK processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: The Rho-associated protein kinase and myosin light chain kinase (ROCK/MYLK) pathway undeniably plays a pivotal role in the pathophysiology of primary open-angle glaucoma (POAG). In our study, we utilized both ocular hypertension (OHT) rabbit models and clinical investigations to gain invaluable insights that propel the development of novel treatments targeting proteins and genes associated with the trabecular meshwork (TM), thereby offering promising avenues for the management of POAG., Methods: Following microbead injections into the anterior chamber of the ocular cavity of rabbits, we observed elevated histiocyte numbers and immune scores for MYLK-4/ pMLC-2, alongside a reduction in the void space within the TM. Notably, treatment was performed with 0.1% ITRI-E-(S)-4046, a compound with dual kinase inhibitor (highly specific inhibitor of ROCK1/2 and MYLK4), significantly reduced intraocular pressure (IOP; P < 0.05) and expanded the void space within the TM (P < 0.0001) compared with OHT rabbits. In clinical investigations, we utilized whole transcriptome sequencing to analyze gene expression specifically related to the TM, obtained from patients (5 early-onset and 5 late-onset) undergoing trabeculectomy., Results: Our findings revealed 103 differential expression genes (DEGs) out of 265 molecules associated with the Rho family GTPase pathway, exhibiting a P value of 1.25E-10 and a z-score of -2.524. These results underscore significant differences between the early-onset and late-onset POAG and highlight the involvement of the ROCK/MYLK pathway., Conclusions: These findings underscore the critical involvement of the ROCK/MYLK pathway in both OHT-related and different onsets of POAG, providing valuable insights into the TM-related molecular mechanisms underlying the disease.

Background: Control of angiogenesis is widely considered a therapeutic strategy, but reliable control methods are still under development. Phosphorylation of myosin light chain 2 (MLC2), which regulates actin-myosin interaction, is critical to the behavior of vascular endothelial cells (ECs) during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP) containing a catalytic subunit PP1. We investigated the potential role of MLC2 in the pharmacological control of angiogenesis., Methods and Results: We exposed transgenic zebrafish Tg(fli1a:Myr-mCherry) ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs., Conclusion: The present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Kiyomi Tsuji-Tamura reports financial support was provided by Japan Society for the Promotion of Science (JSPS). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Background: Tight control of cytoplasmic Ca 2+ concentration in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavβ3, a subunit of voltage-gated Ca 2+ (Cav) channels, in modulating Ca 2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier., Methods: We investigated the function of Cavβ3 in BMECs by Ca 2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavβ3 -/- (Cavβ3-deficient) mice as controls., Results: We identified Cavβ3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavβ3. After induction of experimental autoimmune encephalomyelitis, Cavβ3 -/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavβ3 -/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca 2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca 2+ -dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavβ3 -/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavβ3 -/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavβ3 with inositol 1,4,5-trisphosphate receptor proteins., Conclusions: Independent of its function as a subunit of Cav channels, Cavβ3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca 2+ concentration and Ca 2+ -dependent MLC phosphorylation in BMECs, and this role of Cavβ3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease., Competing Interests: None.

Myosin light chain kinase (MLCK) is a dedicated kinase of myosin regulatory light chain (RLC), playing an essential role in the regulation of muscle contraction and cell motility. Much of the knowledge about MLCK comes from the study of vertebrate MLCK, and little is known about insect MLCK. Here, we identified the single MLCK gene in the locust Locusta migratoria, which spans over 1400 kb, includes 62 exons and accounts for at least five transcripts. We found that the five distinct transcripts of the locust MLCK gene are expressed in a tissue-specific manner, including three muscle-specific isoforms and two generic isoforms. To characterise the kinase activity of locust MLCK, we recombinantly expressed LmMLCK-G, the smallest locust MLCK isoform, in insect Sf9 cells. We demonstrated that LmMLCK-G is a Ca 2+ /calmodulin-dependent kinase that specifically phosphorylates serine 50 of locust muscle myosin RLC (LmRLC). Additionally, we found that almost all LmRLC molecules in the flight muscle and the hindleg muscles of adult locusts are phosphorylated., (© 2024 Royal Entomological Society.)

The mechanism by which aging induces aortic aneurysm and dissection (AAD) remains unclear. A total of 430 participants were recruited for the screening of differentially expressed plasma microRNAs (miRNAs). We found that miR-1204 is significantly increased in both the plasma and aorta of elder patients with AAD and is positively correlated with age. Cell senescence induces the expression of miR-1204 through p53 interaction with plasmacytoma variant translocation 1, and miR-1204 induces vascular smooth muscle cell (VSMC) senescence to form a positive feedback loop. Furthermore, miR-1204 aggravates angiotensin II-induced AAD formation, and inhibition of miR-1204 attenuates β-aminopropionitrile monofumarate-induced AAD development in mice. Mechanistically, miR-1204 directly targets myosin light chain kinase (MYLK), leading to the acquisition of a senescence-associated secretory phenotype (SASP) by VSMCs and loss of their contractile phenotype. MYLK overexpression reverses miR-1204-induced VSMC senescence, SASP and contractile phenotypic changes, and the decrease of transforming growth factor-β signaling pathway. Our findings suggest that aging aggravates AAD via the miR-1204-MYLK signaling axis., (© 2024. The Author(s).)

Background: Inflammation-induced intestinal barrier dysfunction is not only a pathological feature of Crohn's disease (CD) but also an important therapeutic target. Sclareol (SCL) is a nontoxic natural plant compound with anti-inflammatory effect, but its role in CD has not been established., Methods: In vivo studies of mice with TNBS-induced colitis were carried out to evaluate the effects of SCL on CD-like colitis and intestinal barrier function. In vitro, a TNF-α-induced colonic organoid model was established to test the direct effect of SCL on inflammation-induced intestinal barrier injure and inflammatory response. The Nrf2/NF-κB/MLCK signalling was analysed to explore the mechanism of SCL., Results: In vivo, SCL largely alleviated the colitis in TNBS mice, as evidenced by improvements in the weight loss, colitis symptoms, endoscopic score, macroscopic histological score, and histological inflammation score. Moreover, SCL significantly improved intestinal barrier dysfunction, manifested as reduced intestinal permeability and decreased intestinal bacterial translocation in TNBS mice. Importantly, SCL antagonised the intestinal mucosal inflammation while protecting tight junctions in TNBS mice. In vitro, SCL largely depressed pro-inflammatory cytokines levels and improved intestinal epithelial permeability in a TNF-α-induced colonic organoid model. In the context of CD, the protective effects of SCL against inflammation and intestinal barrier damage are at least partially results from the Nrf2 signalling activation and the NF-κB/MLCK signalling inhibition., Conclusions: SCL improved intestinal barrier dysfunction and alleviated CD-like colitis, possibly through modulation of Nrf2/NF-κB/MLCK signalling. In view of SCL's safety profile, there is hope that it will be useful in the clinic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Porcine astrovirus ( PAstV ) has a potential zoonotic risk, with a high proportion of co-infection occurring with porcine epidemic diarrhea virus ( PEDV ) and other diarrheal pathogens. Despite its high prevalence, the cellular mechanism of PAstV pathogenesis is ill-defined. Previous proteomics analyses have revealed that the differentially expressed protein NOD-like receptor X1 (NLRX1) located in the mitochondria participates in several important antiviral signaling pathways in PAstV-4 infection, which are closely related to mitophagy. In this study, we confirmed that PAstV-4 infection significantly up-regulated NLRX1 and mitophagy in Caco-2 cells, while the silencing of NLRX1 or the treatment of mitophagy inhibitor 3-MA inhibited PAstV-4 replication. Additionally, PAstV-4 infection triggered the activation of the extracellular regulated protein kinases/ myosin light-chain kinase (ERK/MLCK) pathway, followed by the down-regulation of tight-junction proteins (occludin and ZO-1) as well as MUC-2 expression. The silencing of NLRX1 or the treatment of 3-MA inhibited myosin light-chain (MLC) phosphorylation and up-regulated occludin and ZO-1 proteins. Treatment of the ERK inhibitor PD98059 also inhibited MLC phosphorylation, while MLCK inhibitor ML-7 mitigated the down-regulation of mucosa-related protein expression induced by PAstV-4 infection. Yet, adding PD98059 or ML-7 did not affect NLRX1 expression. In summary, this study preliminarily explains that NLRX1 plays an important role in the disruption of intestinal mucosal function triggered by PAstV-4 infection via the ERK/MLC pathway. It will be helpful for further antiviral drug target screening and disease therapy.

Dysfunction of tight junction proteins-associated damage to the blood-brain barrier (BBB) plays an important role in the pathogenesis of ischemic stroke. Lifibrate, an inhibitor of cholinephosphotransferase (CPT), has been used as an agent for serum lipid lowering. However, the protective effects of Lifibrate in ischemic stroke and the underlying mechanism have not been clearly elucidated. Here, we employed an in vivo mice model of MCAO and an OGD/R model in vitro . In the mice models, neurological deficit scores and infarct volume were assessed. Evans Blue solution was used to detect the BBB permeability. The TEER was examined to determine brain endothelial monolayer permeability. Here, we found that Lifibrate improved neurological dysfunction in stroke. Additionally, increased BBB permeability during stroke was significantly ameliorated by Lifibrate. Correspondingly, the reduced expression of the tight junction protein ZO-1 was restored by Lifibrate at both the mRNA and protein levels. Using an in vitro model, we found that Lifibrate ameliorated OGD/R-induced injury in human bEnd.3 brain microvascular endothelial cells by increasing cell viability but reducing the release of LDH. Importantly, Lifibrate suppressed the increase in endothelial monolayer permeability and the reduction in TEER induced by OGD/R via the rescue of ZO-1 expression. Mechanistically, Lifibrate blocked activation of the MLCK/ p-MLC signaling pathway in OGD/R-stimulated bEnd.3 cells. In contrast, overexpression of MLCK abolished the protective effects of Lifibrate in endothelial monolayer permeability, TEER, as well as the expression of ZO-1. Our results provide a basis for further investigation into the neuroprotective mechanism of Lifibrate during stroke.

Intestinal barrier integrity is essential for normal nutrient digestion and absorption and disease resistance. This study aims to investigate how fermentation affects the ameliorative effect of bee pollen on the intestinal barrier dysfunction stimulated by interferon-γ and tumor necrosis factor (IFN-γ/TNF-α) cytokines. The results indicated that fermentation enhances the alleviating effect of bee pollen on intestinal barrier dysfunction (including elevated trans epithelial electrical resistance and decreased paracellular permeability). In addition, fermented bee pollen (FBP) significantly decreased (p < 0.05) the secretion levels of interleukin (IL)-6, IL-8, and IL-1β and expression of cyclooxygenase (COX)-2 protein in intestinal barrier cells. Furthermore, fermentation improved the ability of bee pollen to up-regulate the expression of tight junction proteins including zonula occludens (ZO)-1, occluding, and claudin-1. Notably, FBP showed stronger ability to inhibit the expression of nuclear factor kappa-B (NF-κB) mediated myosin light chain kinase (MLCK) and myosin light chain (MLC) signaling pathway associated with phosphorylated proteins. Overall, our results indicated that fermentation enhances the protective effect of bee pollen on the intestinal barrier, and FBP has promising potential to be used as a novel functional food to protect the intestinal barrier., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A deficiency of striated preferentially expressed gene (Speg), a member of the myosin light chain kinase family, results in abnormal myofibril structure and function of immature cardiomyocytes (CMs), corresponding with a dilated cardiomyopathy, heart failure and perinatal death. Mitochondrial development plays a role in cardiomyocyte maturation. Therefore, this study investigated whether Speg deficiency ( - / - ) in CMs would result in mitochondrial abnormalities. Speg wild-type and Speg -/- C57BL/6 littermate mice were utilized for assessment of mitochondrial structure by transmission electron and confocal microscopies. Speg was expressed in the first and second heart fields at embryonic (E) day 7.5, prior to the expression of mitochondrial Na + /Ca2 + /Li + exchanger (NCLX) at E8.5. Decreases in NCLX expression (E11.5) and the mitochondrial-to-nuclear DNA ratio (E13.5) were observed in Speg -/- hearts. Imaging of E18.5 Speg -/- hearts revealed abnormal mitochondrial cristae, corresponding with decreased ATP production in cells fed glucose or palmitate, increased levels of mitochondrial superoxide and depolarization of mitochondrial membrane potential. Interestingly, phosphorylated (p) PGC-1α, a key mediator of mitochondrial development, was significantly reduced in Speg -/- hearts during screening for targeted genes. Besides Z-line expression, Speg partially co-localized with PGC-1α in the sarcomeric region and was found in the same complex by co-immunoprecipitation. Overexpression of a Speg internal serine/threonine kinase domain in Speg -/- CMs promoted translocation of pPGC-1α into the nucleus, and restored ATP production that was abolished by siRNA-mediated silencing of PGC-1α. Our results demonstrate a critical role of Speg in mitochondrial development and energy metabolism in CMs, mediated in part by phosphorylation of PGC-1α., (© 2023. The Author(s).)

Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn's disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2 us ) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The main cause of inflammatory bowel disease (IBD) is abnormal intestinal permeability due to the disruption of the tight junction of the intestinal barrier through a pathogen-mediated inflammatory mechanism and an imbalance of the gut microbiota. This study aimed to evaluate whether 2-ketoglutaric acid alleviated permeability dysfunction with tight junction localization, activated the transforming growth factor beta-activated kinase 1 (TAK1) inflammation pathway, and regulated the homeostasis of the intestinal microbiome in vitro and in vivo IBD model. Our findings revealed that 2-ketoglutaric acid significantly suppressed abnormal intestinal permeability, delocalization of tight junction proteins from the intestinal cell, expression of inflammatory cytokines, such as TNF-α, both in vitro and in vivo. 2-Ketoglutaric acid was found to directly bind to TAK1 and inhibit the TNF receptor-associated factor 6 (TRAF6)-TAK1 interaction, which is related to the activation of nuclear factor kappa B (NF-κB) pathways, thereby regulating the expression of mitogen-activated protein kinase. Dietary 2-ketoglutaric acid also alleviated gut microbiota dysbiosis and IBD symptoms, as demonstrated by improvements in the intestine length and the abundance of Ligilactobacillus, Coriobacteriaceae&#95;UCG&#95;002, and Ruminococcaceae&#95;unclassified in mice with colitis. This study indicated that 2-ketoglutaric acid binds to TAK1 for activity inhibition which is related to the NF-κB pathway and alleviates abnormal permeability by regulating tight junction localization and gut microbiome homeostasis. Therefore, 2-ketoglutaric acid is an effective nutraceutical agent and prebiotic for the treatment of IBD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The physiological importance of cardiac myosin regulatory light chain (RLC) phosphorylation by its dedicated cardiac myosin light chain kinase has been established in both humans and mice. Constitutive RLC-phosphorylation, regulated by the balanced activities of cardiac myosin light chain kinase and myosin light chain phosphatase (MLCP), is fundamental to the biochemical and physiological properties of myofilaments. However, limited information is available on cardiac MLCP. In this study, we hypothesized that the striated muscle-specific MLCP regulatory subunit, MYPT2, targets the phosphatase catalytic subunit to cardiac myosin, contributing to the maintenance of cardiac function in vivo through the regulation of RLC-phosphorylation. To test this hypothesis, we generated a floxed-PPP1R12B mouse model crossed with a cardiac-specific Mer-Cre-Mer to conditionally ablate MYPT2 in adult cardiomyocytes. Immunofluorescence microscopy using the gene-ablated tissue as a control confirmed the localization of MYPT2 to regions where it overlaps with a subset of RLC. Biochemical analysis revealed an increase in RLC-phosphorylation in vivo. The loss of MYPT2 demonstrated significant protection against pressure overload-induced hypertrophy, as evidenced by heart weight, qPCR of hypertrophy-associated genes, measurements of myocyte diameters, and expression of β-MHC protein. Furthermore, mantATP chase assays revealed an increased ratio of myosin heads distributed to the interfilament space in MYPT2-ablated heart muscle fibers, confirming that RLC-phosphorylation regulated by MLCP, enhances cardiac performance in vivo. Our findings establish MYPT2 as the regulatory subunit of cardiac MLCP, distinct from the ubiquitously expressed canonical smooth muscle MLCP. Targeting MYPT2 to increase cardiac RLC-phosphorylation in vivo may improve baseline cardiac performance, thereby attenuating pathological hypertrophy., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Published by Elsevier Inc.)

We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K + concentration in rat caudal arterial smooth muscle involves a Ca 2+ -induced Ca 2+ sensitization mechanism whereby Ca 2+ entry through voltage-gated Ca 2+ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) induced by a Ca 2+ ionophore, ionomycin, rather than by depolarizing stimulation. Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca 2+ . Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of ionomycin-induced 20 kDa light chain of myosin (LC 20 ) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of ionomycin-induced contraction is mediated by MLCK activation by [Ca 2+ ] i elevation, whereas the sustained phase of ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca 2+ ] i elevation.

Long noncoding RNA MYLK antisense RNA 1 (MYLK-AS1) is the crux in multiple diseases. Therefore, the purpose of this study was to investigate the possible mechanism of MYLK-AS1. A total of 62 colon cancer (CC) specimens and paired adjacent normal tissues were collected, and the expression of MYLK-AS1, microRNA (miR)-101-5p/cell division cycle 42 (CDC42) was detected. CC cell lines were transfected with MYLK-AS1, miR-101-5p, CDC42-related plasmids, and the biological functions and markers of epithelial-mesenchymal transition (EMT) were analyzed. The binding relationship between MYLK-AS1, miR-101-5p, and CDC42 was evaluated. In CC tissues and cell lines, MYLK-AS1 and CDC42 were highly expressed, and miR-101-5p was lowly expressed. Inhibition of MYLK-AS1 or upregulation of miR-101-5p can inhibit CC cell growth and EMT. miR-101-5p inhibited CDC42/N-wasp axis activation in CC cells by targeting CDC42. Knockdown of CDC42 or upregulation of miR-101-5p partially reversed the effects caused by upregulation of MYLK-AS1. MYLK-AS1, which is significantly upregulated in CC, may be a molecular sponge for miR-101-5p, and MYLK-AS1 promotes the activation of the CDC42/N-wasp axis in CC cells by targeting CDC42 through miR-101-5p, which in turn promotes tumor development. MYLK-AS1 may be a potential biomarker and target for CC therapy., (© 2023 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Infections like COVID-19 are the primary cause of death around the world because they can cause acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and sepsis. Inflammatory cells serve as crucial protective barriers in these diseases. However, excessive accumulation of inflammatory cells is also one of the major causes of organ damage. The non-muscular myosin light chain kinase (nmMLCK) plays crucial of cytoskeletal components involved in endothelial cell-matrix and cell-cell adhesion, integrity, and permeability. Our previous investigations found that ML-7, a specific inhibitor of MLCK, promoted neutrophil apoptosis through various signaling pathways. In this study, we found that knockout of MLCK significantly promote apoptosis of neutrophils and macrophages in the BALF of the LPS-induced ALI, meanwhile it had no effect on the apoptosis of neutrophils in the circulatory system. RNA-sequencing revealed that the effect of MLCK knockout in inducing apoptosis of inflammatory cells was mediated through lysosomes. Administering ML-7 into the lungs significantly promoted neutrophil apoptosis, accelerating their clearance. In the LPS- or CLP-induced sepsis models, ML-7 administration significantly improves the apoptosis of inflammatory cells, especially neutrophils, at the infection site but had no impact on neutrophils in the circulatory system. ML-7 also significantly improved the survival rate of mice with LPS- or CLP-induced sepsis. Taken together, we found that MLCK plays a crucial role in the survival of inflammatory cells at the infection site. Inhibiting MLCK significantly induces apoptosis of inflammatory cells at the infection site, promoting inflammation resolution, with no impact of the circulatory system., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Calmodulin (CaM) is a universal regulatory protein that modulates numerous cellular processes by using calcium (Ca 2+ ) as the signal. In smooth muscle cells (SMC), one major target of CaM is myosin light chain kinase (MLCK), a kinase that phosphorylates the myosin regulatory light chain and thereby regulates cell contraction. In the absence of CaM, MLCK remains inhibited by its autoinhibitory domain (AID). While it is well established that CaM activates MLCK, the molecular interactions between these two proteins remain elusive due to the lack of structural data. In this work, we constructed a molecular model of mammalian CaM (mCaM) in complex with MLCK leveraging AlphaFold, published biochemical data, and protein-protein docking. The model, along with a strategic set of CaM mutants including a inhibitory variant soybean CaM isoform 4 (sCaM-4), was subject to molecular dynamics (MD) simulations. Using principal component analysis (PCA), we mapped out the transition path for the removal of the AID from the MLCK kinase domain to provide molecular basis of MLCK activation. Additionally , we established MLCK conformations that correspond to the active and inactive states of the kinase. We showed that mCaM and sCaM-4 cause MLCK to undergo the transition to the active and inactive states, respectively. Using two structural metrics, we computed the probabilities of MLCK activation by different CaM variants, which were in good agreement with the experimental data. Distributions along these metrics revealed that different inhibitory CaM variants impair MLCK activation through unique mechanisms. We finally identified molecular contacts that contribute to the MLCK activation by CaM. Overall, we report a de novo molecular model of CaM-MLCK that provides insights into the molecular mechanism of MLCK activation by CaM. The mechanism requires effective removal of the AID while preserving an active configuration of the kinase domain. This mechanism may be shared by other MLCK isoforms and potentially other structurally similar kinases with CaM-mediated regulatory domains.

Bladder cancer and osteosarcoma are 2 types of cancers that originate from epithelial tissues inside the bladder and bone or muscle tissues. Ultrasound-guided biopsies provide crucial support for the diagnosis and treatment of bladder cancer and osteosarcoma. However, the relationship between myosin light chain kinase (MYLK) and caldesmon (CALD1) and bladder cancer and osteosarcoma remains unclear. The bladder cancer datasets GSE65635 and GSE100926, the osteosarcoma dataset GSE39058, were obtained from gene expression omnibus. Differentially expressed genes (DEGs) were screened and weighted gene co-expression network analysis was performed. The construction and analysis of protein-protein interaction network, functional enrichment analysis, gene set enrichment analysis. Gene expression heat map was drawn and immune infiltration analysis was performed. The comparative toxicogenomics database analysis were performed to find disease most related to core gene. Western blotting experiments were performed. TargetScan screened miRNAs that regulated central DEGs. We obtained 54 DEGs. Functional enrichment analysis revealed significant enrichment in terms of cellular differentiation, cartilage development, skeletal development, muscle actin cytoskeleton, actin filament, Rho GTPase binding, DNA binding, fibroblast binding, MAPK signaling pathway, apoptosis, and cancer pathways. Gene set enrichment analysis indicated that DEGs were primarily enriched in terms of skeletal development, cartilage development, muscle actin cytoskeleton, MAPK signaling pathway, and apoptosis. The immune infiltration analysis showed that when T cells regulatory were highly expressed, Eosinophils exhibited a similar high expression, suggesting a strong positive correlation between T cells regulatory and Eosinophils, which might influence the disease progression in osteosarcoma. We identified 6 core genes (SRF, CTSK, MYLK, VCAN, MEF2C, CALD1). MYLK and CALD1 were significantly correlated with survival rate and exhibited lower expression in bladder cancer and osteosarcoma samples compared to normal samples. Comparative toxicogenomics database analysis results indicated associations of core genes with osteosarcoma, bladder tumors, bladder diseases, tumors, inflammation, and necrosis. The results of Western blotting showed that the expression levels of MYLK and CALD1 in bladder cancer and osteosarcoma were lower than those in normal tissues. MYLK and CALD1 likely play a role in regulating muscle contraction and smooth muscle function in bladder cancer and osteosarcoma. The lower expression of MYLK and CALD1 is associated with poorer prognosis., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Bladder cancer (BC) is a malignant tumor that occurs in bladder mucosa. However, relationship between myosin light chain kinase (MYLK) and CALD1 and BC remains unclear. The BC datasets GSE65635 and GSE100926 were downloaded from gene expression omnibus by GPL14951 and GPL14550. Multiple datasets were merged and batched. Differentially expressed genes (DEGs) were screened and weighted gene co-expression network analysis was performed. gene ontology (GO) and Kyoto Encyclopedia of Gene and Genome analysis, gene set enrichment analysis, immune infiltration analysis, survival analysis and Comparative Toxicogenomics Database were performed. TargetScan screened miRNAs that regulated central DEGs. 1026 DEGs were identified. According to GO analysis, DEGs were mainly enriched in cancer pathway, cGMP-PKG signaling pathway, Apelin signaling pathway and proteoglycans in cancer. The enrichment items are similar to GO and Kyoto Encyclopedia of Gene and Genome enrichment projects for DEGs, which were mainly enriched in cancer pathways and leukocyte trans-endothelial cell migration. Among enrichment projects of metascape, GO has regulation of the enzyme-linked receptor protein signaling pathway and silk-based process, as well as an enrichment network stained by enrichment terms and P values. Nine core genes (ACTA2, MYLK, MYH11, MYL9, ACTG2, TPM1, TPM2, TAGLN and CALD1) were obtained, which were highly expressed in tumor tissue samples and lowly expressed in normal tissue samples. Nine genes were associated with necrosis, inflammation, tumor, edema, and ureteral obstruction. MYLK and CALD1 are highly expressed in the BC. The higher expression of MYLK and CALD1, the worse prognosis., Competing Interests: The authors have no funding and conflicts of interest to disclose.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Spinal cord injury (SCI) can prompt an immediate disruption to the blood-spinal cord barrier (BSCB). Restoring the integrity of this barrier is vital for the recovery of neurological function post-SCI. The UTX protein, a histone demethylase, has been shown in previous research to promote vascular regeneration and neurological recovery in mice with SCI. However, it is unclear whether UTX knockout could facilitate the recovery of the BSCB by reducing its permeability. In this study, we systematically studied BSCB disruption and permeability at different time points after SCI and found that conditional UTX deletion in endothelial cells (ECs) can reduce BSCB permeability, decrease inflammatory cell infiltration and ROS production, and improve neurological function recovery after SCI. Subsequently, we used RNA sequencing and ChIP-qPCR to confirm that conditional UTX knockout in ECs can down-regulate expression of myosin light chain kinase (MLCK), which specifically mediates myosin light chain (MLC) phosphorylation and is involved in actin contraction, cell retraction, and tight junctions (TJs) protein integrity. Moreover, we found that MLCK overexpression can increase the ratio of p-MLC/MLC, further break TJs, and exacerbate BSCB deterioration. Overall, our findings indicate that UTX knockout could inhibit the MLCK/p-MLC pathway, resulting in decreased BSCB permeability, and ultimately promoting neurological recovery in mice. These results suggest that UTX is a promising new target for treating SCI., (© 2023. The Author(s).)

Endometriosis is initiated by the movement of endometrial cells in the uterus to the fallopian tubes, the ovaries and the peritoneal cavity after the shedding of the uterus lining. To cause endometriosis, it is often necessary for these endometrial cells to migrate, invade and grow at the secondary site. In the present study, immortalized human endometriosis stromal cells (HESC) were employed to look for the inhibitors of migration and invasion. Using a chemical library of bioactive metabolites, it was found that an NF‑κB inhibitor, DHMEQ, inhibited the migration and invasion of HESC. Both whole‑genome array and metastasis PCR array analyses suggested the involvement of myosin light chain kinase (MLCK) in the mechanism of inhibition. DHMEQ was confirmed to inhibit the expression of MLCK and small inhibitory RNA knockdown of MLCK reduced cellular migration and invasion. The addition of DHMEQ to the knockdown cells did not further inhibit migration and invasion. DHMEQ is particularly effective in suppressing disease models by intraperitoneal (IP) administration and this therapy is being developed for the treatment of inflammation and cancer. DHMEQ IP therapy may also be useful for the treatment of endometriosis.

Intestinal barrier dysfunction often exists in the heat stroke (HS) pathological process, which increases intestinal permeability and induces endotoxemia. The upregulation of MLCK is a crucial player affecting intestinal permeability. This study aimed to explore whether inhibiting myosin light chain kinase (MLCK) can improve HS-induced intestinal injury in rats. Twelve-week-old Wistar male rats were divided into three groups: the control group, the HS model group, and the treatment group [HS model + ML-7 (MLCK inhibitor)]. HS impaired the tight junctions in the rat gut and increased permeability. Additionally, increased inflammatory factors in serum, activation of apoptosis, and downregulation of tight junction proteins were observed in intestinal cells. ML-7 significantly inhibited the MLCK/p-MLC2 signaling pathway, increased the expression of tight junction proteins, reduced intestinal permeability, reduced apoptosis and alleviated the intestinal damage caused by HS. ML-7 inhibited HS-induced apoptosis of intestinal epithelial cells by regulating the ERK/p38/HSP70 axis. Furthermore, inhibition of MLCK upregulated HSP70 expression through activation of the ERK pathway and inhibited cell apoptosis by abolishing the p38 MAPK pathway. In conclusion, inhibiting the MLCK/p-MLC2 signaling pathway reduces HS-induced intestinal permeability and protects the intestinal mucosal barrier., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the most fatal human malignancies worldwide. In this research, we aimed to identify long noncoding RNAs (lncRNAs) as biomarkers for HCC diagnosis and prognosis. lncRNA expression profiles were obtained from Gene Expression Omnibus and The Cancer Genome Atlas databases. The differentially expressed lncRNAs between HCC and adjacent tissues were analyzed with bioinformatic tools. Four lncRNAs with area under the curve of the receiver operating characteristic curve >0.9 were selected from both datasets. Univariate and Kaplan-Meier analyses were performed to obtain LINC01093 , MYLK-AS1 , and MCM3AP-AS1 as the optimal diagnostic and prognostic biomarkers. Finally, qPCR confirmed that LINC01093 and MYLK-AS1 were significantly differentially expressed in HCC and adjacent normal tissues. In general, we demonstrated that novel lncRNAs, LINC01093 and MYLK-AS1 , could be used as potential diagnostic and prognostic biomarkers for HCC.

Background: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3 , regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure., Methods: We generated the knock-in mice ( Mylk3 +/fs and Mylk 3 fs/fs ) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation ( MYLK3 +/fs ) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154)., Results: Both mice ( Mylk3 +/fs and Mylk 3 fs/fs ) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9&#95; MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3 +/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9&#95; MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the V max for ventricular myosin regulatory light chain phosphorylation without affecting the K m . LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3 +/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3 / PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes., Conclusions: cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure., Competing Interests: Disclosures None.

Fluoride is a common contaminant of groundwater and agricultural commodity, which poses challenges to animal and human health. A wealth of research has demonstrated its detrimental effects on intestinal mucosal integrity; however, the underlying mechanisms remain obscure. This study aimed to investigate the role of the cytoskeleton in fluoride-induced barrier dysfunction. After sodium fluoride (NaF) treatment of the cultured Caco-2 cells, both cytotoxicity and cytomorphological changes (internal vacuoles or massive ablation) were observed. NaF lowered transepithelial electrical resistance (TEER) and enhanced paracellular permeation of fluorescein isothiocyanate dextran 4 (FD-4), indicating Caco-2 monolayers hyperpermeability. In the meantime, NaF treatment altered both the expression and distribution of the tight junction protein ZO-1. Fluoride exposure increased myosin light chain II (MLC2) phosphorylation and triggered actin filament (F-actin) remodeling. While inhibition of myosin II by Blebbistatin blocked NaF-induced barrier failure and ZO-1 discontinuity, the corresponding agonist Ionomycin had effects comparable to those of fluoride, suggesting that MLC2 serves as an effector. Given the mechanisms upstream of p-MLC2 regulation, further studies demonstrated that NaF activated RhoA/ROCK signaling pathway and myosin light chain kinase (MLCK), strikingly increasing the expression of both. Pharmacological inhibitors (Rhosin, Y-27632 and ML-7) reversed NaF-induced barrier breakdown and stress fiber formation. The role of intracellular calcium ions ([Ca 2+ ] i ) in NaF effects on Rho/ROCK pathway and MLCK was investigated. We found that NaF elevated [Ca 2+ ] i , whereas chelator BAPTA-AM attenuated increased RhoA and MLCK expression as well as ZO-1 rupture, thus, restoring barrier function. Collectively, abovementioned results suggest that NaF induces barrier impairment via Ca 2+ -dependent RhoA/ROCK pathway and MLCK, which in turn triggers MLC2 phosphorylation and rearrangement of ZO-1 and F-actin. These results provide potential therapeutic targets for fluoride-induced intestinal injury., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Inflammatory bowel disease (IBD) is continuously increasing globally and caused by intestinal barrier dysfunction. Although protocatechuic acid (PCA) has a protective effect on colitis, the molecular mechanisms underlying its contribution to intestinal barrier function remain unknown. Transepithelial electrical resistance (TEER) and FITC-dextran permeability measurements reveled that PCA suppresses lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α-induced increase in intestinal permeability; zonula occludens (ZO)-1 and claudin-2 redistribution was also suppressed in the epithelial cell membranes of differentiated Caco-2 cells. PCA was found to directly bind Rho-associated coiled-coil containing protein kinase (ROCK), subsequently suppressing myosin light chain (MLC) phosphorylation. Notably, PCA binds ROCK to a similar degree as Y27632, a selective ROCK inhibitor. Orally administering PCA (5 or 25 mg per kg per day) to C57BL/6 mice alleviated the 3% dextran sulfate sodium (DSS)-induced colitis symptoms including reduced colon length, disrupted intestinal barrier structure, and increased proinflammatory cytokines expressions, such as interleukin (IL)-1β, TNF-α, and IL-6. Furthermore, orally administering PCA suppressed DSS-induced ZO-1 and claudin-2/4 redistribution in mice colon membrane fractions. Therefore, PCA may serve as a promising nutraceutical to improve gut health and alleviate IBD by maintaining intestinal barrier function in vitro and in vivo .

Aims: Aging impairs cardiac function and increases susceptibility to myocardial ischaemic injury. Cardiac myosin light chain kinase (MLCK3) phosphorylates cardiac myosin regulatory light chain (MLC2), controlling sarcomere organization and cardiomyocyte contraction. Dysregulation of MLCK3 and phosphorylated MLC2 (p-MLC2) contributes to heart failure after myocardial infarction (MI). We aimed at exploring how the MLCK3-p-MLC2 axis changes in aging hearts post MI and at investigating the underlying regulatory mechanisms., Methods and Results: We generated adult (3 months) and aged (30 months) MI mouse models to compare their cardiac performance, and then detected MLCK3 expression and MLC2 activity. Aging increased the size of MI-induced infarctions and promoted cardiac contractile dysfunction. Furthermore, MLCK3 expression and MLC2 activity increased in adult hearts after MI, but not in aged hearts. miR-146a was found consistently increased in adult and aged hearts post MI. Mechanistic analyses performed in vitro demonstrated that miR-146a-5p down-regulated matrix metalloprotease (MMP)2/16 expression in cardiomyocytes. This down-regulation in turn increased MLCK3 expression and MLC2 activity. However, miR-146a-5p failed to regulate the MMP2/16-MLCK3-p-MLC2 axis in senescent cardiomyocytes or in cardiac miR-146a conditional knockout mice, with the latter experiencing an exacerbated deterioration of cardiac function post MI., Conclusion: These results suggest that an increase of MLCK3 and p-MLC2 contents through decreasing MMP2/16 by miR-146a-5p represents a compensatory mechanism that can protect cardiac contractile function after MI. Aging impairs this miR-146a-5p-regulated MMP2/16-MLCK3-p-MLC2 contractile axis, leading to compromised contractile function and increased susceptibility to heart failure., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Objective: Intestinal barrier loss is a Crohn's disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically., Design: Protein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects., Results: MLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects., Conclusion: Binding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions., Competing Interests: Competing interests: WVG is a founder, shareholder and employee of Thelium Therapeutics. JRT is a founder and shareholder of Thelium Therapeutics and has served as a consultant for Entrinsic, Immunic, and Kallyope., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Intestinal epithelial barrier function is closely associated with the development of many intestinal diseases. Heat-killed Lacticaseibacillus paracasei (HK-LP) has been shown to improve intestinal health and enhance immunity. However, the function of HK-LP in the intestinal barrier is still unclear. This study characterized the inflammatory effects of seven HK-LP (1 μg/mL) on the intestinal barrier using lipopolysaccharide (LPS) (100 μg/mL)-induced Caco-2 cells. In this study, HK-LP 6105, 6115, and 6235 were selected, and their effects on the modulation of inflammatory factors and tight junction protein expression (claudin-1, zona occludens-1, and occludin) were compared. The effect of different cultivation times (18 and 48 h) was investigated in response to LPS-induced intestinal epithelial barrier dysfunction. Our results showed that HK-LP 6105, 6115, and 6235 improved LPS-induced intestinal barrier permeability reduction and transepithelial resistance. Furthermore, HK-LP 6105, 6115, and 6235 inhibited the pro-inflammatory factors (TNF-α, IL-1β, IL-6) and increased the expression of the anti-inflammatory factors (IL-4, IL-10, and TGF-β). HK-LP 6105, 6115, and 6235 ameliorated the inflammatory response. It inhibited the nuclear factor kappa B (NF-κB) signaling pathway-mediated myosin light chain (MLC)/MLC kinase signaling pathway by downregulating the Toll-like receptor 4 (TLR4)/NF-κB pathway. Thus, the results suggest that HK-LP 6150, 6115, and 6235 may improve intestinal health by regulating inflammation and TJ proteins. Postbiotics produced by these strains exhibit anti-inflammatory properties that can protect the intestinal barrier.

Aquafeeds are susceptible to contamination caused by aflatoxin B1 (AFB1). The gill of fish is an important respiratory organ. However, few studies have investigated the effects of dietary AFB1 exposure on gill. This study aimed to discuss the effects of AFB1 on the structural and immune barrier of grass carp gill. Dietary AFB1 increased reactive oxygen species (ROS) levels, protein carbonyl (PC) and malondialdehyde (MDA) contents, which consequently caused oxidative damage. In contrast, dietary AFB1 decreased antioxidant enzymes activities, relative genes expression (except MnSOD) and the contents of glutathione (GSH) (P < 0.05), which are partly regulated by NF-E2-related factor 2 (Nrf2/Keap1a). Moreover, dietary AFB1 caused DNA fragmentation. The relative genes of apoptosis (except Bcl-2, McL-1 and IAP) were significantly upregulated (P < 0.05), and apoptosis was likely upregulated through p38 mitogen-activated protein kinase (p38MAPK). The relative expressions of genes associated with tight junction complexes (TJs) (except ZO-1 and claudin-12) were significantly decreased (P < 0.05), and TJs were likely regulated by myosin light chain kinase (MLCK). Overall, dietary AFB1 disrupted the structural barrier of gill. Furthermore, AFB1 increased gill sensitivity to F. columnare, increased Columnaris disease and decreased the production of antimicrobial substances (P < 0.05) in grass carp gill, and upregulated the expression of genes involved with pro-inflammatory factors (except TNF-α and IL-8) and the pro-inflammatory response partly attributed to the regulation by nuclear factor κB (NF-κB). Meanwhile, the anti-inflammatory factors were downregulated (P < 0.05) in grass carp gill after challenge with F. columnare, which was partly attributed to the target of rapamycin (TOR). These results suggested that AFB1 aggravated the disruption of the immune barrier of grass carp gill after being challenge with F. columnare. Finally, the upper limit of safety of AFB1 for grass carp, based on Columnaris disease, was 31.10 μg/kg diet., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Cardiac contraction is modulated by the phosphorylation state of myosin regulatory light chain 2 (MLC-2v). The level of MLC-2v phosphorylation is dependent on the opposing activities of MLC kinases and phosphatases. The predominant MLC phosphatase found in cardiac myocytes contains Myosin Phosphatase Targeting Subunit 2 (MYPT2). Overexpression of MYPT2 in cardiac myocytes results in a decreased level of MLC phosphorylation, reduced left ventricular contraction, and induction of hypertrophy; however, the effect of knocking out MYPT2 on cardiac function is unknown. We obtained heterozygous mice containing a MYPT2 null allele from the Mutant Mouse Resource Center. These mice were produced in a C57BL/6N background which lack MLCK3, the main regulatory light chain kinase in cardiac myocytes. We found that mice null for MYPT2 were viable and had no obvious phenotypic abnormality when compared to WT mice. Additionally, we determined that WT C57BL/6N mice had a low basal level of MLC-2v phosphorylation, which was significantly increased when MYPT2 was absent. At 12-weeks, MYPT2 KO mice had smaller hearts and showed downregulation of genes involved in cardiac remodeling. Using cardiac echo, we found that 24-week-old male MYPT2 KO mice had decreased heart size with increased fractional shortening compared to their MYPT2 WT littermates. Collectively, these studies highlight the important role that MYPT2 plays in cardiac function in vivo and demonstrate that its deletion can partially compensate for the lack of MLCK3., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Advanced glycation end products (AGEs) disturb endothelial barrier function and contribute to age-related diseases. As microRNAs (miRNAs) are potential therapeutic agents, targeting AGEs-associated signaling using miRNAs in endothelial cells may be an effective intervention strategy for age-related vascular disorders. This study investigated the effects of AGEs on the endothelial cell senescence and barrier function in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with AGEs and transfected with miRNA-1-3p mimics to induce overexpression of miR-1-3p. Senescence-associated β-galactosidase (SA-β-Gal) staining and senescence-related proteins P53, P21, and P16 were detected to evaluate the endothelial cell senescence. The expression levels of myosin light chain kinase (MLCK) signaling and transendothelial electric resistance (TEER) were used to indicate endothelial barrier function. AGEs significantly increased SA-β-gal staining-positive cells accompanied by the upregulation of P53, P21, and P16 expression. AGEs also damaged endothelial barrier function by decreasing TEER and increasing zonula occludens protein 1, p-MLC/MLC, and MLCK. miRNA-1-3p was significantly reduced in HUVECs treated with AGEs. miR-1-3p overexpression decreased MLCK signal and improved AGEs-induced endothelial barrier function impairment. Meanwhile, miR-1-3p overexpression ameliorated oxidative stress and endothelial cell senescence induced by AGEs. AGEs induced endothelial cell senescence and endothelial barrier dysfunction by regulating miR-1-3p/MLCK signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Pulmonary arterial hypertension (PAH) is characterized by endothelial dysfunction, uncontrolled proliferation and migration of pulmonary arterial endothelial cells leading to increased pulmonary vascular resistance resulting in great morbidity and poor survival. Bone morphogenetic protein receptor II (BMPR2) plays an important role in the pathogenesis of PAH as the most common genetic mutation. Non-muscle myosin light chain kinase (nmMLCK) is an essential component of the cellular cytoskeleton and recent studies have shown that increased nmMLCK activity regulates biological processes in various pulmonary diseases such as asthma and acute lung injury. In this study, we aimed to discover the role of nmMLCK in the proliferation and migration of pulmonary arterial endothelial cells (HPAECs) in the pathogenesis of PAH. We used two cellular models relevant to the pathobiology of PAH including BMPR2 silenced and vascular endothelial growth factor (VEGF) stimulated HPAECs. Both models demonstrated an increase in nmMLCK activity along with a robust increase in cellular proliferation, inflammation, and cellular migration. The upregulated nmMLCK activity was also associated with increased ERK expression pointing towards a potential integral cytoplasmic interaction. Mechanistically, we confirmed that when nmMLCK is inhibited by MLCK selective inhibitor (ML-7), proliferation and migration are attenuated. In conclusion, our results demonstrate that nmMLCK upregulation in association with increased ERK expression may contribute to the pathogenesis of PAHby stimulating cellular proliferation and migration.

Introduction: The progression of placental diseases such as preeclampsia is closely related to trophoblast dysfunction. Recent studies indicated the dysregulation of N6-methyladenosine (m 6 A) RNA modification in trophoblast disorders, while the function of METTL3, a methyltransferase of m 6 A, in trophoblasts remains to be studied., Methods: The expression of METTL3 was determined by real-time PCR and immunoblotting. METTL3 expression in trophoblast cell lines HTR-8/SVneo and JEG-3 was knocked down using shRNA. The invasion of trophoblast cells in Matrigel was determined using xCELLigence. The m 6 A-containing transcripts was determined by m 6 A-sequencing in HTR-8/SVneo cells. The myosin light chain kinase (MYLK) gene was transfected into HTR-8/SVneo cells., Results: The expression of METTL3 was downregulated in preeclamptic placentae compared to normal placentae. Knockdown of METTL3 repressed the invasion of extravillous trophoblast cells. Mechanistically, METTL3 promoted the stability of MYLK mRNA through m 6 A modification. Overexpression of MYLK rescued retarded cell invasion by METTL3 depletion., Discussion: Collectively, our results highlight an essential role of METTL3-MYLK axis in trophoblast invasion., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: The intestinal tract is considered the body's "engine" and the most impacted organ during sepsis. In this study, we explored toll-like receptor 4 (TLR4) functions in sepsis-induced intestinal barrier dysfunction., Methods: Wild-type and TLR4-knockout (KO) mice were used to establish a sepsis-induced dysfunctional intestinal barrier model via the intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). Hematoxylin and eosin staining, Transmission electron microscope, enzyme linked immunosorbent assay, western blot, quantitative real-time polymerase chain reaction, TdT-mediated dUTP nick end labeling staining, 16 S rRNA gene sequencing were used to explore differences in inflammatory cytokines, apoptosis, tight junction (TJ) protein expression, and intestinal flora diversity between groups., Results: TLR4-deficiency reduced procalcitonin and C-reactive protein to prevent sepsis, and also inhibited inflammatory response by decreasing interleukin (IL)- 1β, IL-6 and tumor necrosis factor-α levels. Also, BAX/Bcl 2 and cleaved-caspase 3 expressions were decreased in TLR4-KO mice to suppress the intestinal mucosal cell apoptosis. TJ proteins, including zonula occludens protein, Occludin and Claudin-5 were significantly increased and intestinal fatty acid binding protein, myosin light chain and myosin light chain kinase were reduced in TLR4-KO mice. Additionally, 16 S rRNA gene sequencing indicated that TLR4-deficiency improved flora diversity and altered normal and abnormal bacterial proportions., Conclusions: TLR4 deficiency alleviated LPS-induced intestinal barrier dysfunction by reducing inflammatory responses and apoptosis, impairing intestinal damage, and regulating intestinal flora disturbance., Competing Interests: Conflict of interest statement The authors declare that they have no competing interests., (Copyright © 2022. Published by Elsevier Masson SAS.)

L. plantarum 1.0386 repairs intestinal epithelial tight junction injury, and the present study was designed to further explore the role of its postbiotics, including the surface protein (1.0386-Slp), peptidoglycan (1.0386-PG) and exopolysaccharide (1.0386-EPS). The results showed that they all could improve the lipopolysaccharide (LPS)-induced decrease of transepithelial electrical resistance, increase of paracellular permeability, release of inflammatory factors, and disruption of tight junctions in Caco-2 cells, and the repairing effect of 1.0386-Slp was better than those of 1.0386-PG and 1.0386-EPS, and was similar to that of L. plantarum 1.0386. Moreover, either L. plantarum 1.0386 or 1.0386-Slp intervention significantly increased the expression of miR-200c inhibited by LPS, while the miR-200c inhibitor weakened the ability of 1.0386-Slp to promote the expression of tight junction proteins (ZO-1, occludin and claudin-1). Meanwhile, 1.0386-Slp restored the distribution of tight junction proteins and inhibited the increase of NF-κB p65, MLC and pMLC protein expression evoked by LPS. However, the addition of miR-200c inhibitors or mimics weakened or strengthened the down-regulation of MLCK-MLC pathway protein expression by 1.0386-Slp, respectively. In summary, 1.0386-Slp may be the main efficacy component of L. plantarum 1.0386, and miR-200c may be involved in the process of 1.0386-Slp inhibiting the MLCK pathway to repair intestinal epithelial tight junction injury.

Zearalenone (ZEN) is a widespread contaminant of cereals and agricultural products which causes food safety issues. Ingesting food or feed contaminated with ZEN can disrupt the intestinal epithelial barrier function. The RhoA/ROCK signaling pathway plays a key role in regulating the epithelial barrier function, but studies on such roles have rarely focused on the intestine. The aim of this experiment was to investigate the exact mechanism of ZEN-induced intestinal barrier damage and whether the RhoA/ROCK signaling pathway is involved. The results showed that ZEN significantly induced alkaline phosphatase (AP) activity and FITC-dextran (4 kDa) passage across the epithelial barrier, which significantly reduced the transepithelial resistance (TEER). Meanwhile, ZEN could induce the significantly down-regulated mRNA expression of tight junction proteins (occludin, claudin-1, ZO-1, and claudin-3) and redistribution of ZO-1 immunofluorescence. Further studies demonstrated that ZEN exposure activated the RhoA/ROCK signaling pathway, significantly up-regulated the mRNA expression of ROCK1, the main effector of the signaling pathway, the protein expression of phosphorylated myosin light chain (MLC) and myosin light chain kinase (MLCK), and relatively increased the activity of ATP in cells, simultaneously remodeling the cytoskeleton (F-actin). Overall, our study indicated that ZEN induced intestinal barrier dysfunction by activating the RhoA/ROCK signaling pathway.

Scope: The consumption of artificial sweeteners has been rapidly increasing, with potentially hazardous effects on human reproduction. This study aims to explore the effect of Acesulfame Potassium (Ace K) and its potential mechanism to induce uterine contraction through in vitro, ex vivo, in vivo, and clinical observation studies., Methods and Results: Used ex vivo and in vitro studies to analyze its effect on uterine contraction and involved signaling pathway. Used the long-term, high-dose exposure to examine Ace K's affection for contractive-related protein expression. By involving a cohort of 613 participants, to assess the dose-responsiveness of Ace K consumption and calculate the odd ratio of Ace K consumption and the relationship with preterm risk. Animal studies show increasing uterine contraction, cytokine secretion, and altered contraction-related protein expression. Human data show that higher consumption of Ace K may be related to early delivery., Conclusion: Long-term high-dose exposure to Ace K can induce uterine hypercontraction, increase cytokine secretion, and alters contraction-related protein expression. These findings suggest that women who suffer from uterine hypercontraction causes painfulness should pay more attention to the zero- or low-calorie soft drinks or food products containing Ace K., (© 2022 Wiley-VCH GmbH.)

Bifidobacteria are important mediators of immune system development within the gastrointestinal system and immunological homeostasis. The present study explored the anti-colitic activity of Bifidobacterium bifidum H3-R2 in a murine dextran sulfate sodium (DSS)-induced model of ulcerative colitis (UC). Moreover, this study offers novel insight regarding the molecular basis for the probiotic properties of B. bifidum H3-R2 by analyzing the underlying mechanisms whereby B. bifidum H3-R2-derived proteins affect the intestinal barrier. B. bifidum H3-R2 administration was sufficient to alleviate clinical manifestations consistent with DSS-induced colitis, restoring aberrant inflammatory cytokine production, enhancing tight junction protein expression, and positively impacting overall intestinal microecological homeostasis in these animals. Moreover, the bifidobacteria-derived GroEL and transaldolase (TAL) proteins were found to regulate tight junction protein expression via the NF-κB, myosin light chain kinase (MLCK), RhoA/Rho-associated protein kinase (ROCK), and mitogen-activated protein kinase (MAPK) signaling pathways, preventing the lipopolysaccharide (LPS)-mediated disruption of the intestinal epithelial cell barrier.

Aims: This study aims to investigate whether 3,4-dihydroxyphenylacetic acid (DHAA) can improve gut barrier function by inhibiting the mitogen-activated protein kinase (MAPK) - myosin light-chain kinase (MLCK) signaling pathway in type 2 diabetes (T2D) mice., Main Methods: T2D mice were induced by a high-fat diet combined with streptozotocin. T2D mice were intragastrically administered with DHAA at 75 and 150 mg kg/body weight per day for 4 weeks. Blood glucose, insulin level, oxidative stress and inflammatory cytokines were measured. TJ (tight junction) protein and MAPK-MLCK pathway-related proteins were analyzed by western blot., Key Findings: DHAA alleviated hyperglycemia and decreased insulin resistance of T2D mice. It also decreased oxidative stress via increased glutathione (GSH) and total superoxide dismutase (T-SOD) activities and reduced containing malondialdehyde (MDA). DHAA exhibited a significant anti-inflammatory effect by decreasing the level of pro-inflammatory cytokines lipopolysaccharide (LPS) and interleukin (IL)-6 and increasing that of anti-inflammatory cytokine IL-10. More importantly, DHAA improved gut barrier function by enhancing tight junction protein expression and inhibiting the MAPK-MLCK signaling pathway., Significance: DHAA could reduce oxidative stress, decrease inflammatory response, and improve intestinal function in T2D mice, which may help to relieve the symptoms of T2D., (Copyright © 2022 Elsevier Inc. All rights reserved.)